Stroke is a pathological reduction in blood flow which causes irreversible damage of brain tissue, long-term disruptions of brain functions, and frequently patient death. The major focus of ischemia research is on the mechanisms of cell death and the search for pharmacological approaches to salvage vulnerable neural cells. Substantially less attention is paid to long-term impairment of neural cell function that is not associated with cell death. Existing literature suggest, nonetheless, that even relatively mild ischemia causes long-lasting suppression of synaptic communication. For unknown reasons, only presynaptic release is affected, while postsynaptic responses remain preserved. In the current study we hypothesize that suppression of synaptic transmission occurs due to nitrosation of cysteine residues in N-ethylmaleimide sensitive fusion protein (NSF) by nitric oxide (NO) and NO-related reactive nitrogen species. The NSF protein is a trimeric ATPase which is crucial for continuous synaptic vesicle docking/fusion. Modification of a critical cysteine residues in the NSF by N-ethylmaleimide causes irreversible inhibition of vesicle docking and fusion. Similarly, NOdependent modification of NSF thiols may cause long-term inhibition of vesicular neurotransmitter release. To test our hypothesis we propose the following specific aims. (1) We will test for the increased modification of thiols in the ischemic tissue up to 24 hours after ischemia and will further demonstrate thiol modification in the NSF protein immunoprecipitated from the ischemic brain. (2) Using synaptosomal preparation we will test whether nitric oxide and related reactive nitrogen species modify NSF and via this mechanism inhibit vesicular neurotransmitter release. We will further attempt to reverse NSF nitrosation and defects in neurotransmitter release by applying thiol reducing agents. This project will clarify the molecular mechanisms of less understood long-term disruption of synaptic transmission in ischemia and may suggest additional approaches for patient treatment and rehabilitation after stroke and transient ischemic attacks. [unreadable] [unreadable] [unreadable]